1. Technical Field
The invention relates generally to homogeneous preparations of polyoximes and to preparations of hetero-polyoximes, said polyoximes being macromolecules of defined structure containing a plurality of oxime linkages, and to reagents and methods for assembly of such molecules.
2. Background
Two methods have traditionally been used to produce complex polymers such as polypeptides. One method relies on relatively uncontrolled polymerization reactions, wherein monomer subunits react to produce large polymers. Using this method, polydisperse macromolecules such as polypeptides and plastics (e.g., polyethylene or nylon) can be produced from monomeric residues such as amino acids or small aliphatic or aromatic organic molecules, respectively. While such polydisperse macromolecules can be relatively easy to produce, the polymeric macroscopic products are not homogeneous at the molecular level but are mixtures of polymers of different lengths and even different composition, e.g., in a random copolymer. Furthermore, the similarity of the homologs produced in such polydisperse preparations makes it difficult or impossible to obtain a single high molecular weight product in pure form.
The second method utilized to obtain complex macromolecules has been the sequential assembly of reversibly protected monomers. This approach can be used to obtain products of defined, typically linear, structure. Unfortunately, the method is limited in the size and, most critically, the complexity of molecules that can be produced. For example, synthesis of defined polypeptides or proteins larger than about 50-80 amino acid residues has been beyond the reach of this technology. Condensation of pre-purified protected peptides, two at a time, is limited by xe2x80x9cthe insolubility of large protected fragments. As a result, synthesis of homogeneous, linear polypeptides, for example, is limited to an upper limit of about 100 amino acid residues. Mutter et al. (Proteins: Structure, Function and Genetics (1989) 5:13-21) have synthesized branched chain polypeptides by step-wise coupling of protected amino acids to a synthetic, protected, resin-bound peptide template during solid-phase peptide synthesis. Deprotection and cleavage was required to obtain a soluble template-assembled synthetic protein. Also using step-wise, solid phase peptide synthesis, Tam and Zavala (J. Immunol. Meth. (1989) as multiple antigen peptides, which were subsequently obtained in soluble, crude form after HF deprotection and cleavage.
Since protecting groups used in polypeptide synthesis generally decrease solubility of the protectable molecule, the ability to condense unprotected polypeptides would provide an improvement in the solubility problem encountered using protected precursors, as well as minimize harsh deprotection methods needed to achieve a final product. However, the use of unprotected precursors raises the seemingly insurmountable problem of regiospecificity. Therefore, attempts have been made to use regiospecific condensation of unprotected fragments through the use of chemoselective ligation.
Chemoselective ligation requires the use of complementary pairs of reactive groups present at specific sites on the precursor molecules that are being joined. The use of reactive groups having complementary chemical reactivity, such as a thiol group and a bromoacetyl group, results in the formation of a bond in a regiospecific manner. For example, thiol-type chemoselective ligations have been used to prepare multi-antigenic peptides. In an attempt to avoid harsh deprotection methods and formation of impure products caused by possible steric hindrance between closely spaced growing peptide arms during step-wise solid phase synthesis, Drijfhout and Bloemhoff (Int. J. Peptide Protein Res. (1991) 37:27-32) used thiol-type chemoselective coupling by synthesizing a branched xe2x80x9cocta-amino lysine treexe2x80x9d peptide, whose deprotected amino groups were extended to contain protected sulfhydryl groups (S-acetylmercaptoacetyl) for subsequent coupling to an appropriately modified sulfhydryl-containing antigenic peptide. However, the product obtained had poor characteristics as defined by high performance liquid chromatography and was not fully characterized. More recently, thiol chemistry was used to prepare, in a two-fragment condensation, a totally synthetic, linear, functional HIV protease analog (Schnolzer and Kent (1992) Science 256:221-225).
Unfortunately, thiol chemistry is not completely specific. It is well known, for example, that thiol groups can participate in disulfide bond shuffling. In addition, alkylating agents such as bromoacetyl and maleoyl can react with nucleophilic amino acid groups other than a thiol group. For example, bromoacetyl can react with the thioether side chain of methionine residues, thus limiting the homogeneity and/or complexity of design of the desired product.
Thus, a need exists for preparations, particularly homogeneous preparations, of easily synthesized macromolecules of defined structure having stable ligation linkages and for reagents and methods for constructing these preparations of macromolecules that provide ease, rapidity and mildness of synthesis; essentially quantitative yields; versatility in design; and applicability to construction using a diversity of biochemical classes of compounds. In addition a need exists for macromolecules, particularly homogeneous macromolecules, of defined structure that can be designed for desired activity, solubility, conformation and other desirable properties; that present components, such as peptides or oligonucleotides, in non-linear, polyvalent form; that provide higher binding affinity and specificity of interaction; and, in the case of homogeneous macromolecules, can made available as homogeneous preparations. Furthermore the need exists for libraries of macromolecules such as peptides or oligonucleotides having advantages discussed herein. The present invention satisfies these needs and provides related advantages as well.
The present invention is directed to homogeneous preparations of easily synthesized, homogeneous macromolecules and to easily synthesized heterogenous macromolecules, said macromolecules being of defined structure and containing a plurality of stable oxime linkages and to reagents and methods for rapid and specific assembly of such macromolecules by chemoselective ligation via oxime formation.
The macromolecules of defined structure comprise an organic molecule (referred to as a baseplate) to which other molecules (referred to as COSMs) will be attached having a plurality of oxime linkages, preferably at least three, to a plurality of a second organic molecule (COSM). In the case of homopolyoxime macromolecules the COSMs are identical to each other. In one embodiment the oxime linkages are in the same orientation. In the case of hetero-polyoxime macromolecules, the baseplate is attached to a plurality of second organic molecules where at least one of the second organic molecules attached to the baseplate is different from the other attached second organic molecules. Also provided are two reagents for constructing these molecules: baseplates having a plurality of oxime-forming reactive groups and a second organic molecule having an orthogonal reactive group complementary in oxime linkage formation to the oxime forming orthogonal reactive groups present on the baseplate. For the purposes of the present invention, the second organic molecules are alternatively referred to as complementary orthogonal specifically active molecules (xe2x80x9cCOSMxe2x80x9d) and are defined further below. The oxime linkages provide a hydrolytically stable means of joining oligomer or macromolecule subunits. The various reactive groups are referred to herein as xe2x80x9corthogonalxe2x80x9d groups, which means that they are complementary to each other in reactivity and do not react with other functional groups present in the precursors of the macromolecule being formed.
Also provided by this invention are methods of preparing these molecules, including by parallel self-assembly, comprising chemoselectively linking each of a plurality of orthogonal reactive groups on a baseplate to its complementary reactive orthogonal group present on one of a plurality of a second organic molecule via oxime bond formation. The baseplate and second organic molecules are preferably formed from amino acid resides. In other embodiments, baseplates and/or second organic molecules are formed from or further comprise sugar residues, nucleic acid residues and/or lipids. Preferred baseplates are prepared by controlled, organic chemical syntheses, such as solid phase peptide synthesis (xe2x80x9cSPPSxe2x80x9d) or the equivalent for nucleic acids or carbohydrate-containing baseplates. Carbohydrate-containing baseplates are preferably isolated from natural or recombinant sources or prepared enzymatically. Homogeneous preparations of isolated peptides or proteins (prepared for example by recombinant means) are also suitable for use in making baseplate embodiments; however, such molecules must be designed or chosen to contain residues compatible with subsequent regio-specific modification to obtain a plurality of oxime-forming, orthogonal reactive groups, and, in addition, maintain a defined structure and significant homogeneity after the regio-specific modification. Accordingly, heterogeneous antibody glycoprotein preparations subsequently modified by chemical or enzymatic oxidation of sugar residues are not suitable baseplate embodiments of the present invention.
Methods of using these polyoxime compounds are also provided, including use as antigens and immunogens. Compositions, including pharmaceutical compositions, comprising these compositions of matter are provided. Kits providing polyoximes of the invention or reagents for their synthesis are provided.
The polyoxime compounds of the invention are relatively complex, synthetically prepared macromolecules. The most massive species prepared to date is an approximately 20 kD complex, containing 195 amino acid residues: the homo-polyoxime octa-oxime formed between NH2OCH2CO-ELGGGPGAGSLQPLALEGSLQKR-OH. (SEQ ID NO: 10) and Oxe2x95x90CHxe2x80x94CO-Gly3-[Lys(COCHO)]7-Gly-OH (SEQ ID NO: 9). As will be apparent to those skilled in the art in the light of this disclosure, even larger and more complex molecules are now readily accessible in homogeneous and defined form given the rapid, specific, high-yielding, mild and versatile methods of preparing these compounds provided by this invention.